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IGFBP-rP1 induces p21 expression through a p53-independent pathway, leading to cellular senescence of MCF-7 breast cancer cells

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Abstract

Objectives

Insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1), a member of the IGFBP super family, was identified as a potent tumor suppressor in several carcinomas. IGFBP-rP1 was down-regulated in primary breast cancer tissues and several breast cancer cell lines but overexpressed in senescent human mammary epithelial cells (HMECs), suggesting that IGFBP-rP1 might be a tumor suppressor in breast cancer and the tumor suppressor role of IGFBP-rP1 might be associated with cellular senescence. The aim of the study was to observe the effect of IGFBP-rP1 on cellular senescence and the molecular events mediating this biological effect in MCF-7 breast cancer cells.

Methods

DNA fragment-encoding IGFBP-rP1 was cloned in-frame N-terminally to EGFP gene to generate IGFBP-rP1-EGFP fusion protein expression plasmid (pEGFP-IGFBP-rP1). The plasmid pEGFP-IGFBP-rP1 was then transfected into MCF-7 cells, and the proliferation, cell cycle distribution, cellular senescence, and cell cycle-related protein expression of MCF-7 cells were examined by trypan blue exclusion, flow cytometry, senescence-associated galactosidase (SA-β-gal) staining, and Western blot analysis, respectively. Two shRNA plasmid vectors against p21 or p53 gene were constructed and stably transfected into the MCF-7 cells to determine the involvement of p21 or p53 in cellular senescence induced by IGFBP-rP1.

Results

Transfection of IGFBP-rP1 or addition of condition medium (CM) from IGFBP-rP1-transfected cells in MCF-7 cells caused induction of a variety of senescent phenotypes, such as decrease in cell proliferation, increase in G0/G1 cell cycle arrest cells, change in cell morphology, and increase in senescence-associated galactosidase (SA-β-gal) activity. IGFBP-rP1-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21 and dephosphorylation of the retinoblastoma protein (pRB). Cell proliferation block and cellular senescence induction in response to IGFBP-rP1 were partially reversed by p21 knockdown in MCF-7 cells. Knockdown of p53 in MCF-7 cells did not influence the growth inhibition, cellular senescence, and p21 expression of the cells in response to IGFBP-rP1 transfection.

Conclusions

Results from this study suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 enhanced expression, which regulated through the p53-independent pathway. IGFBP-rP1 might be one of the key molecules that trigger cellular senescence in breast cancer. Restoration of IGFBP-rP1 function might have therapeutic significance in breast cancer.

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Acknowledgments

We thank Dr. Xicai Wang (Tumor Institute of Yunnan Province, Tumor Hospital of Yunnan Province, the Third Affiliated Hospital of Kunming Medical University) and Dr. Wenhai Yan (Department of Pathophysiology, Basic Medicine School, Zhengzhou University) for the technical assistance and suggestions. This work was supported by the Innovation Group Foundation Grant of Luohe Science and Technology Bureau (No. LK2010-46-13-1) and by the College Foundation of Luohe Medical College (No. LHMC2010-01).

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Authors and Affiliations

  1. Experimental Center of Molecular Medicine, Luohe Medical College, No. 148 University Road, 462002, Luohe, Henan Province, China

    Shuguang Zuo, Chang Liu, Jianguo Wang, Fuqing Wang, Wanling Xu, Shao Cui, Lei Yuan, Xudong Chen, Wenjuan Fan, Mingchen Cui & Guohua Song

  2. School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, China

    Shao Cui

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  1. Shuguang Zuo
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  2. Chang Liu
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Correspondence to Mingchen Cui or Guohua Song.

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Shuguang Zuo, Chang Liu, and Jianguo Wang contributed equally to this work.

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Zuo, S., Liu, C., Wang, J. et al. IGFBP-rP1 induces p21 expression through a p53-independent pathway, leading to cellular senescence of MCF-7 breast cancer cells. J Cancer Res Clin Oncol 138, 1045–1055 (2012). https://doi.org/10.1007/s00432-012-1153-y

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